Current systems and methods for collection, extraction, and detection of nucleic acid from biological samples for testing are typically complicated, requiring multiple steps with technically trained personnel, and not optimized for processing samples with large volumes or for preventing cross-contamination in sample processing and stabilization for shipment.
A variety of body fluids, such as blood, plasma, serum, Cerebrospinal fluid (CSF), pleural effusion, ascites, urine etc., contain short chain nucleic acid (NA) fragments, namely, cell-free nucleic acids (cfNA), or circulating nucleic acids (cNA). Altered nucleic acids, originated endogenously from a tumor, or “exogenously” from fetus or pathogenic infection inside the body, may present as cfNA in the peripheral blood at very low concentrations and may be detectable, and further, be distinguishable from normal host cfNA. Extraction of sufficient amount of those cfNA from plasma or serum for testing requires processing a relatively large volume of fluid, which imposes a unavoidable technical challenge in clinical diagnostic settings. Accordingly, there is a need the field for new methods to meet such challenges.
Exemplary such methods for detecting, for example, tuberculosis, are described in Pending PCT published application WO2012135815, invented by the inventor of this application, and incorporated herein by reference. Such testing, however, may be most useful in regions of the world lacking ready access to the expensive processing equipment used in analysis of the samples. Accordingly, there is a need in the art for a collection system and methodology that will permit capturing nucleic acid in sufficient amounts from large volume biological samples, to run later analysis, to prevent contamination from the environment and operators, and to preserve and ship the nucleic acid, so that nucleic acid can be collected at a point of care facility using relatively inexpensive equipment, and then shipped in a stabilized form to a central location for further processing and assays.
Various methods of extraction suitable for isolating circulating DNA or RNA from large volumes of biological fluids are known, such as those described, for example, in QIAamp® Circulating Nucleic Acid Handbook, (2nd edition, 02, 2011, Qiagen), and an improved spin column extraction method described in U.S. Pat. No. 8,685,742, based on technological principles from U.S. Pat. No. 5,234,809 (Boom technology). U.S. Pat. No. 5,346,994 describes a technology an organic liquid extraction method using phenol-chloroform. Both of these methods may be used for large volume extraction, such as from plasma or serum specimens, but the organic reagents are toxic, which limits its use. Both of the technologies mentioned above require a high-speed centrifuge (>10,000 G), though elution or precipitation, to obtain the extracted NA for downstream applications.
Also, U.S. Pat. Nos. 7,897,378 and 8,158,349 describe devices and method for purifying or isolating nucleic acids from larger sample volumes, including systems comprising a pair of cooperating hollow bodies through which samples are passed into a collection vessel, with nucleic acids bound to a binding material in one of the hollow bodies. The hollow body containing the retained sample is transferred to a first receiving vessel for washing, then the purified or isolated nucleic acids are eluted and collected in a second receiving vessel for further analysis. Again, a high-speed centrifuge is required for eluting bound nucleic acids from the solid phase matrix.
U.S. Pat. No. 5,234,809 (Boom), for example, incorporated herein by reference, discloses a method for isolating nucleic acids, which is suitable for a multiplicity of different uses. It describes a method for isolating nucleic acids from nucleic acid-containing starting materials by incubating said starting material with a chaotropic buffer and a DNA-binding solid phase. The chaotropic buffers effect, if necessary, both lysis of the starting material and binding of the nucleic acids to the solid phase.
A novel biomolecule extraction system was first described by the Applicant in U.S. Provisional Patent Application Ser. No. 61/827,244 and PCT Application Ser. No. US14/39320 (“the Original Applications”), both of which are incorporated herein by reference in their entireties. The Original Applications disclosed inventions relating to methods and systems for processing a biological sample, namely lysing, binding, washing, stabilizing and eluting biomolecules of the biological sample. One embodiment included a system for collecting a sample of nucleic acid, the system comprising a receptacle defining an internal volume, a removable cap for the receptacle and having a connection interface in fluid communication with a sample connection port in the cap, a filter column adapted to be removably attached to the connection interface of the receptacle cap, a sample collection container, and a shipping container. Various other components of the system were disclosed. The Original Applications also disclosed a method for collecting a sample of nucleic acid, the method comprising the steps of: (a) providing the collection system described herein; (b) collecting a volume of sample-containing fluid in the sample collection container; (c) connecting the sample collection container to the receptacle via the sample collection port; (d) passing the volume of sample-containing fluid from the sample collection container through the filter column, thereby collecting the sample on the substrate and collecting a remainder in the receptacle; (e) placing the shipping container open end over the filter column, engaging the filter column with the shipping container, and detaching the filter column from the receptacle cap and (f) temporarily sealing the shipping container with the removable lid. The disclosed methods are particularly useful for processing the collected nucleic acid at minimally-equipped medical-care settings, such as small, remote and/or peripheral clinics, and shipping the collected samples to a better-equipped central laboratory for further analysis of the collected samples for detection of a disease, such as for detection of latent tuberculosis.
As molecular technologies rapidly advance, biomarker detection of circulating cell-free nucleic acids (NA) including cell-free DNA and cell-free RNA (cfDNA and cfRNA) respectively, or together (cfNA or cNA) in plasma, serum and other body fluids is emerging as less invasive means for diagnosis and prognosis of prenatal genetic abnormalities, cancers, solid organ transplantation rejection and infectious diseases such as tuberculosis, from early discoveries. However, due to extremely low abundance in body fluids, cfNA as clinical analyte is still facing various technical issues that affect cfNA sample quality, quantity, and consequently the final diagnostic results. Technical issues include: 1) sample collection/transportation, 2) sample processing, and 3) the potential opportunity using cfRNA for disease status monitoring.
Sample Collection/Transportation Issues
Most accessible sources of cfNA are plasma or serum (together PS) of peripheral blood. Plasma concentrations of cfNA in normal individuals are very low, in the range of 1.8-44 ng/ml or about 500-10000 genome equivalents/ml (ge/ml). The trace amount of tumor-derived cfNA or circulating tumor NA (ctNA) in PS, if exists, may be merely one to a few hundred copies per ml, or about 0.005-0.01% of total cfNA (4). In order to collect sufficient target ctNA, a large volume of PS, i.e. 1-5 ml is often needed. In addition, release of even small percentage of cellular DNA or RNA from blood will cause difficulties in downstream analysis of the targeted cfNA. To prevent cfNA degradation and genomic NA (gNA) release from blood cells, separation of the PS from blood cells should typically take place within 2 hours, 2-4 hours or within 7 hours after phlebotomy based on different protocols. Separation usually requires 1 step or 2 steps of centrifugation at 1000-2000 g for 10 minutes and optionally, 5000-16000 g. Separation of serum by simple centrifugation after blood clotting may be easier, but may introduce a predictable degree of cell lysis due to clotting that may not significantly affect final analysis. Though the concentrations of targeted ctDNA in plasma and serum are about the same, it is noticed that serum contains more large gDNA fragments, possibly released from blood cells during clotting.
Another issue of sample collection is temperature. The PS after separation from blood cells typically needs to be stored at −20 C (for short time) or −80 C (for long time). Sample collection sites such as phlebotomy sites are not always in close proximity to the molecular diagnostic facility. Thus, cryopreservation of frozen PS during transportation typically needs to be maintained. Novel blood collection devices, Cell-Free DNA BCT™ (BCT) and Cell-Free RNA BCT, were developed by Streck Inc. (NE). They prevent cellular DNA and RNA release and stabilize cfDNA and cfRNA in blood at ambient temperature for up to 7 days and 2 days respectively. The BCT technology partially solves the issues of separation delay and the transportation condition. However, it still faces the obstacles of sample processing to be performed in the molecular diagnostic facility, as described below.
Sample Processing Issues
The clinical applications of cfNA testing are also hindered by sample processing, i.e., efficient extraction, enrichment, and recovery of trace fragile cfNA from large volume of same fluids. Nucleic acid extraction and purification from biological materials is generally based on two approaches: organic extraction and solid phase absorption. The organic extraction, namely, guanidine thiocyanate-phenol/chloroform method, is applicable for large volume of biological fluids such as PS, but not as suitable for use in a clinical laboratory, due to toxicity of reagents and multiple hands-on processing steps. Solid phase extraction, based on Boom technology, has been progressively developed into two major formats: column (or spin column) and magnetic bead technologies. The basic principle is that high concentrated chaotropic agents disrupt second and third structures of proteins and lipid complexes, inactivate enzymes-including DNA and RNA nucleases, and release nucleic acids from bound microstructures (Lysing). Adding alcohol into the lysate facilitates binding of the free nucleic acids to an absorbent matrix (Binding). In the column (spin column) format, a lysate-binding mixture is added into a micro-column and flows through porous matrix (i.e., silica membrane) by centrifugation or vacuum. DNA or RNA in the mixture is absorbed on the matrix and the rest (waste) is removed. Then, 1-2 step(s) of washing are typically performed to remove residue contaminant (Washing). Finally, the bound nucleic acids are released from the matrix and are collected to a new tube by centrifugation (Eluting). Automation of spin column operation is not easy; however, an automated instrument (QIAcube) specifically for spin columns has been developed recently (Qiagen). Processing a sample with a spin column typically requires four to five times repeated centrifugation, such as with a high speed desktop centrifuge. With a vacuum apparatus, the centrifugation may be reduced to one step (elution), but multiple pipettings are still necessary. Spin columns are generally designed to process small volume samples, often <300 μl. A popular kit, QIAamp® DNA Blood Mini kit (DBM, Qiagen) has been extensively used for fetal cfDNA or ctDNA extraction from maternal blood. The volume capacity of spin columns is believed to limit its use in cfNA extraction for sensitivity-demand studies.
Another specifically designed kit for cfNA extraction is Qiagen's QIAamp® circulating Nucleic acid kit (Q-CNA kit), which comprises an extension tube, as described in U.S. Pat. No. 8,685,742, the contents of which are hereby incorporated by reference. A Q-CNA kit features several advantages: 1) it has extendable capacity for the sample volume (1-5 ml), 2) it is vacuum-enabled, and, particularly 3) it is formulated for recovery of short cfNA fragments in PS. In a recent stringent comparison study, it was found recovery of short DNA (115 pb and 461 bp) with Q-CNA kit is 3-4 times more than that with Qiagen's DBM kit (24). Though Q-CNA kit has been adopted with increasing popularity in cfNA extraction for quantitative PCR (qPCR), digital PCR (dPCR), and next generation sequencing (NGS) applications in tumor diagnosis and non-invasive prenatal test (NIPT), there are still several issues to prevent it from being widely used in clinical setting: 1) multiple-repeated pipetting steps raise the question of possible mis-pipetting and cross-contamination; 2) the Q-CNA is configured for use with a vacuum manifold QIAvac™ 24 Plus (Qiagen) powered by a vacuum pump, so in operation, the sample tubes are exposed to air and subjected to alternating negative pressure; therefore, contamination from environment may occur; 3) furthermore, inconsistent flow rate and occasionally lysate-binding mix clogs on the porous silica membrane may cause uneven flow and/or may completely block flow-through; 4) finally, to elute the solid-phase-bound CNA, a high speed centrifugation is still inevitable. Thus, all above mentioned methods require certain equipment and electricity supply.
Silica coated magnetic bead (MB) technology is based on the same principle and has similar steps as described above. Unlike the column format in which the absorbent is fixed in the column, magnetic beads are dispensed into the lysate and collected with magnetic field. Operation of MB processing is relatively easy due to automation. Several manufacturers provide multiple models tailored to specific needs, but, most of them have a sample volume capacity limited to 1 ml.
Thus, a need remains for systems and methods addressing one or more of the foregoing issues, and that improve upon and providing alternative embodiments to the collection systems described in the Original Applications.